Von Recklinghausen Neurofibromatosis (NF-1) is a common inherited disease of humans; patients with NF-1 can develop a variety of symptoms, the most common of which are peripheral nerve tumors made up largely of Schwann cells and extracellular matrix. Neurofibromas develop sporadically in NF-1 patients, suggesting that mutations in the NF-1 gene cause only a predisposition to formation of tumors, which are then triggered by additional, or "secondary" genetic or epigenetic events. We have defined several cellular abnormalities in neurofibroma cells. To understand the sequence of events which leads to neurofibroma formation, we will test whether each of these abnormalities is a direct consequence of the NF-1 mutation and therefore is observed in non-tumor-related NF-1 peripheral nerves (first-hit related) or is a result of second hits and observed only in tumors. Overproliferation of Schwann cells is diagnostic of neurofibroma formation, and Schwann cells from neurofibromas require mitogenic stimulation of proliferate in vitro. These observations suggest that accumulation of mitogenic activities in NF-1 tumors may be important in stimulating Schwann cell proliferation. We have detected and partially purified mitogenic activity from neurofibroma which appears to be related to neuron-derived growth factor (NDGF), an axon-associated mitogen which we have purified to homogeneity from fetal calf brain. Excessive proliferation of Schwann cells could result from alterations in the structure and activity of mitogen or aberrant expression. In fact, NDGF in normal nerves is associated only with particulate fractions, but in neurofibromas a significant proportion of mitogenic activity is soluble. We will purify neurofibroma mitogen to homogeneity and prepare specific antibodies which recognize mitogen. We will compare neurofibroma mitogen to NDGF and use immunological methods to determine mitogen distribution in normal and NF-1 tissue. We will test whether Schwann cells in neurofibromas proliferate only near sources of mitogen. Epigenetic factors suggested to promote tumor formation (second hits) in vivo will be tested to determine whether they stimulate proliferation of NF-1 non-neuronal cells in vitro assays, or modulate response of NF-1 cells to mitogen. These experiments will identify steps in formation of neurofibromas which could be useful in the development of clinical treatment of NF-1.